Nef is an HIV-1 accessory factor essential for viral pathogenesis and immune escape of HIV- infected cells. Many Nef functions require self-association (dimerization), and small molecules that interfere with Nef dimerization may represent a new approach to HIV therapeutics. In this application, which is responsive to PAR-12-058 (Solicitation of Assays for High Throughput Screening to Discover Chemical Probes), we propose to use a cell-based assay for Nef dimerization to discover chemical probes for Nef function that may also represent new antiretroviral drug leads. In this assay, Nef is fused to non-fluorescent, complementary fragments of YFP and co-expressed in the same cell population. Dimerization of Nef results in juxtaposition of the YFP fragments and reconstitution of the fluorophore, a process known as bimolecular fluorescence complementation (BiFC). To simplify the assay for automation, the two Nef-YFP fusion proteins plus an mRFP reporter are expressed from a single vector that drives equivalent translation of all three proteins from a single transcript. Validation studies revealed that Nef-BiFC/mRFP ratios resulting from cells expressing wild-type Nef vs. a dimerization-defective Nef mutant were very clearly separated, with Z-factors consistently in the 0.6-0.7 range. Fully automated pilot screens of ~3,000 compounds identified several active structures that reproducibly blocked Nef dimerization in the low micromolar range. Secondary assays showed that these compounds bind directly to Nef and inhibit its functions related to HIV infectivity and receptor down- regulation. Here we propose to screen a large collection of diverse chemical compounds and evaluate their activity against this critical HIV virulence factor with the following Specific Aims: 1. Screen a large, diverse chemical collection for inhibitors of Nef dimerization using a cell-based BiFC approach. We will implement the Nef-BiFC assay for fully automated screening of 60,000 discrete compounds from two complementary libraries. These include 10,000 non-peptide peptidomimetics from ChemDiv designed to block diverse protein-protein interfaces and 50,000 structures selected from the ChemBridge 410,000 compound core library to maximize structural diversity while enhancing predicted physiochemical properties. 2. Perform follow-up assays to identify the most selective and potent cell-active inhibitors of Nef dimerization, and explore thei mechanism of action. This Aim will identify the most potent and selective chemical probes of HIV-1 Nef function. The first group of assays will determine whether or not each hit compound interacts directly with the Nef protein and impacts its self-association in vitro and in silico, whle the second assay set will explore antiretroviral activity and mechanism in cell-based systems. Successful completion of this project will bring us closer to our long-term goal of discovering cell-active compounds that selectively and potently inhibit as many HIV-1 Nef functions as possible.